Blocking Buffers for Protein Immunoassays

Blocking buffers and reagents to support your immunoassay development including blocking, stability, and detection reagents.

Blocking buffers and reagents play an important role in immunoassay-based research, where they keep proteins in their native state outside of their natural biological environment.

Abcam offers a variety of blocking, stability and detection reagents as well as TMB (3,3’,5,5’-Tetramethylbenzidine). TMB is a common chromogenic substrate for HRP detection. Different kinetic formulations of TMB are available for the optimization of different applications.

Which Kinetic TMB Formulation Should be Chosen?

The type of kinetic TMB substrate determines the dynamic range and assay sensitivity of an ELISA experiment:

  • Provide an accurate standard curve
  • Improve the sensitivity and capabilities of an assay
  • Allow for low protein concentrations to be detected over short time periods

Table 1: Range of TMB substrates with different kinetic formulations.

Product Function Product code
TMB ELISA substrate (Highest Sensitive) Most sensitive TMB formulation ab171522
TMB ELISA substrate (High Sensitivity) Most popular and robust formulation ab171524
TMB ELISA substrate (Slow Kinetic Rate) Allow a wide dynamic/working range without compromising the assay sensitivity ab171525
TMB ELISA substrate (Slower Kinetic Rate) Option for maximum dynamic/working range ab171526
TMB ELISA substrate (Slowest Kinetic Rate) Option for extended dynamic range and improved reproducibility ab171527

A plot of the different kinetics for various TMB formulations after a sandwich ELISA experiment where the HRP conjugated detection antibody was diluted with HRP Stability Conjugate Reagent (ab171537) and the capture antibody was blocked with Immunoassay Blocking Buffer (ab171534).

Figure 1. A plot of the different kinetics for various TMB formulations after a sandwich ELISA experiment where the HRP conjugated detection antibody was diluted with HRP Stability Conjugate Reagent (ab171537) and the capture antibody was blocked with Immunoassay Blocking Buffer (ab171534).

The Role of Blocking Buffers

Blocking buffers are used to preserve the shape, native folded state, and conformational arrangement of proteins either when in solution, or when dried, to ensure that the proteins remain biologically active. This ensures that antibodies recognize the proteins and can attach.

Table 2: Buffers to preserve the conformational spatial arrangement of proteins.

Product Stabilization ELISA WB Micro-spheres
Immunoassay Blocking Buffer ab171534 x x x
Immunoassay Blocking and Protein Stabilizer (BSA free) ab171535 x x x x
AP Stability Conjugate Reagent ab171536 x
HRP Stability Conjugate Reagent ab171537 x

Data from comparative studies using buffers with various antibodies (monoclonal and polyclonal) can are below:

Following 30 months at room temperature in Immunoassay Blocking Buffer (ab171534) at, 70% of monoclonal antibody activity, and 95% of polyclonal antibody activity, were retained.

Figure 2. Following 30 months at room temperature in Immunoassay Blocking Buffer (ab171534) at, 70% of monoclonal antibody activity, and 95% of polyclonal antibody activity, were retained.

Following 30 months at room temperature in Immunoassay Blocking and Protein Stabilizer (BSA free) (ab171535), greater than 85% of monoclonal antibody activity and greater than 90% of polyclonal antibody activity were retained.

Figure 3. Following 30 months at room temperature in Immunoassay Blocking and Protein Stabilizer (BSA free) (ab171535), greater than 85% of monoclonal antibody activity and greater than 90% of polyclonal antibody activity were retained.

Table 3: Blocking and conjugate buffers guide.

Product Function
Immunoassay Blocking Buffer - ab171534 Blocking agent that can be dried down or used for same-day assays. Preserves the conformation and activity of dried antibodies, proteins or enzymes coated to a surface
Immunoassay Blocking Buffer (BSA free) - ab171535 Blocking solution without BSA. Synthetic formulation to eliminate background issues
AP Stability Conjugate Reagent - ab171536 Preserves AP conjugated proteins in native folded state in solution
HRP Stability Conjugate Reagent - ab171537 Preserves HRP conjugated proteins in native folded state in solution

About Abcam

Abcam is a global life sciences company providing highly validated antibodies and other binders and assays to the research and clinical communities to help advance the understanding of biology and causes of disease.

Abcam’s mission is to serve life scientists to help them achieve their mission faster by listening to their needs, continuously innovating and improving and by giving them the tools, data and experience they want. Abcam’s ambition is to become the most influential life science company for researchers worldwide.


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Last updated: Apr 1, 2019 at 6:01 AM

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